How amino acids control the binding of Cu(II) ions to DNA. Part III. A novel interaction of a histidine complex with DNA

L-Histidine Cu(II) complex bound to DNA showed broad EPR signals characteristic of the aggregated Cu(II) species, which could be observed even when the molar ratio of L-histidine to Cu(II) ion was smaller than unity. The signal for the DNA fibers changed with the orientation of the fibers in the static magnetic field. Based on these results, the signal was assigned to a mono-histidine Cu(II) complex stereospecifically aggregated in a groove or along a phosphodiester chain of the double helical DNA. In contrast to the L-histidine complex, the D-histidine complex bound to DNA did not show such broad signals and the observed spectra for the complex on B-form DNA fibers at -150 degrees C were simulated assuming that the g1 axis of the mono-D-histidine complex tilts by about 55 degrees from the DNA-fiber axis. Addition of some deoxy-nucleotides, but not deoxy-nucleosides, to a solution of a mono-histidine complex resulted in the formation of a dinuclear ternary complex with different structures for L- or D-histidine, suggesting the possibility that the stereospecific aggregation of the L-histidine complex on a double helical DNA was mediated by the phosphodiester backbones.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.     

Structural organization of copper(II) ions in complexes with DNA

The interaction of Cu(II) ions with native and denatured DNA as a function of ionic strength of the solution was studied by the equilibrium dialysis method. Graphical analysis of binding isotherms confirmed the occurrence of interstrand and intrastrand binding of Cu(II) with DNA and made possible determination of the respective binding constants. To facilitate interpretation of the data, a new molecular model of Cu(II)-DNA binding has been proposed, assuming interstrand intercalation of one Cu(II) ion between two GC pairs both in the successive even and odd groups of GC pairs, and interstrand binding of Cu(II) to the isolated GC pairs, with the exception of T-C-T and T-G-T sequences. In agreement with this model, the DNA-Cu(II) complex is most stable under the equilibrium with free Cu(II) ions at 4 degrees C, pH 6 when the molar ratio of GC pairs to Cu(II) ions bound interstrandially attains GC/Cuinter = 2 +/- 0.1.     

The DNA-bound orientation of Cu(II)-Xaa-Gly-His metallopeptides

The DNA-bound orientations of Cu(II) x Xaa-Gly-L-His metallopeptides (where Xaa is Gly, L-Lys or L-Arg) were investigated by DNA fiber EPR spectroscopy and molecular modeling. Observed and calculated EPR spectra indicated that the g// axes of 1:1 Cu(II) complexes of the tripeptides tilted about 50 degrees from the DNA fiber axis. These results suggest that the complexes are stereospecifically oriented in the DNA minor groove. Although the side chain of the N-terminal amino acid residue did not affect the orientation of the DNA-bound complexes, it contributed to their stability in the presence of DNA; the Cu(II) complex of Gly-Gly-L-His was found to dissociate to hydrated Cu(II) ion more extensively than the respective L-Lys-Gly-L-His and L-Arg-Gly-L-His complexes. The ionic interaction between the positively charged lysine or arginine residues and the negatively charged DNA phosphodiester backbone may result in the reduced dissociation of these complexes when bound to the DNA minor groove.     

DNA cleavage study using copper (II)-GlyAibHis: a tripeptide complex based on ATCUN peptide motifs

Development of new chemical nucleases is a matter of great interest because of their extensive use in biotechnology and as therapeutic agents. The ATCUN (amino terminal Cu(II) and Ni(II) binding) is a peptide motif that occurs naturally in the serum albumins. The similar peptide motif (GlyAibHis) having unnatural amino acid Aib (alpha-aminoisobutyric acid) was synthesized and its Cu(II) complex was characterized by ESI-MS and spectrophotometry studies. The reactivity of this complex toward DNA cleavage has been investigated. Cu(II)-GlyAibHis shows the DNA cleavage only in presence of mild oxidizing agents like ascorbate by an oxidative mechanism rather than hydrolytic and follows the pseudo first order kinetics (K obs = 0.085 min(-1)). The non-hydrolytic mechanism was further supported by the hydrolysis of pNPP which followed the pseudo first order kinetics (K obs = 1.98 x 10(-2) min(-1)) having no pH effect.     

Synergetic DNA-cleaving activities of the metal complexes of a polyether-tethered pyrrole-polyamide dimer

A simple polyether-tethered pyrrole-polyamide dimer 1 was synthesized in 50% yield from the reaction of 2,2,2-trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethanone with 2,2'-[1,2-ethanediylbis(oxy)]bisethanamine, and fully characterized on the basis of 1H- and 13C-NMR, MS, HR-MS, and IR data. Agarose gel-electrophoresis study of the cleavage of plasmid pBR322 DNA by the complexes of compound 1 with seven metal ions indicated that most of the metal complexes were capable of efficiently cleaving DNA at pH?7.0 and 37°. Among them, the Cu(II) complex exhibited the highest activity, with the maximal catalytic rate constant k(max) and Michaelis constant K(M) being 5.61?h?1 and 7.30?mM, respectively. Spectroscopic, ESI-MS, ethidium-bromide (EB) displacement, and viscosity experiments indicated that compound 1 could form a 1?:?1 complex with Cu(II) ion, and that this complex showed moderate binding affinity toward calf-thymus DNA.     

Specific nickel(II)-transfer process between the native sequence peptide representing the nickel(II)-transport site of human serum albumin and L-histidine.

The kinetics and mechanism for Ni(II)-transfer of the native sequence tripeptide, L-aspartyl-L-alanyl-L-histidine-N-methylamide (AAHNMA), representing the Ni(II)-transport site of human serum albumin (HSA) and L-histidine (L-His) was studied in forward and reverse reactions in the pH range 6.5 to 9.0 at I = 0.2 and 25 degrees C. For the Ni(II)-transfer from Ni(II)-(L-His)2 to native sequence peptide, the rate-determining step is the formation of a mixed-ligand complex of NiH-1AB by deprotonation of peptide nitrogen from NiAB where A and B denote the anionic forms of AAHNMA and L-His, respectively. For the Ni(II)-transfer from Ni(II)-peptide to L-His, the rate-determining step is a bond breaking between Ni(II) and peptide nitrogen to form NiH-1A by protonation to a peptide nitrogen of NiH-2A. The equilibrium constants for the metal-transfer reaction of MH-2A + 2HB in equilibrium MB2 + A (A = Ni(II), Cu(II] were 10(3.29) and 10(0.78) for Ni(II) and Cu(II), respectively. NiB2 is 324 times as stable as CuB2. Furthermore, the ratio of Ni(II)/Cu(II) in the rate constants for the reaction of MB2 with A was found to be 2.8 x 10(-4). Thus, despite the similarities of Cu(II) and Ni(II) in the metal-binding sites of HSA and in reaction mechanism, Ni(II)-(L-His)2 complex is so stable thermodynamically and kinetically, compared to the Cu(II)-(L-His)2 complex, that Ni(II) is hardly transferred from Ni(II)-(L-His)2 to native sequence peptide. These findings may support specificities in the Ni(II)-transfer, its organ distribution, and its excretion through urine in vivo.     

A solution study of the interaction of the Cu(II) ions with HisGlyGlyTrp tetrapeptide and its evaluation as superoxide dismutase mimetic complex

The superoxide anion radical is a highly reactive toxic species produced during the metabolic processes. A number of copper (II) complexes with amino acids and peptides are known to show superoxide dismutase (SOD) like activity. The design and application of synthetic low molecular weight metal complexes as SOD mimics have received considerable attention during the last decade. A variety of di- and tri-peptides containing histidyl residue in different positions have been employed to bind Cu(II) and to show the activity. But reports on Cu(II) complex with tetra-peptide having histidine amino acid in this regard are limited. As the HGGGW peptide having His at its N-terminal is reported to be a potential moiety for Cu(2+) binding, in the present work the synthesis of HisGlyGlyTrp peptide and its complexation with copper (II) ions has been reported. The interaction of synthesized peptide with Cu(II) was studied by electron spray ionization-mass spectrometer (ESI-MS) and UV-Vis spectroscopic methods. The species distribution was studied by combined spectrophotometric and potentiometric methods. The studies were performed at 25 ± 0.1 °C with constant ionic strength (μ = 0.1 M NaNO(3)) in aqueous solution using Bjerrum-Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The solution studies suggested that the pH of the medium play important role in the different species formation of the copper complexes. Species distribution curves indicate that Cu complexation takes place at all physiological pH values from 3-11. The resultant copper (II) peptide complex at physiological pH was tested for superoxide dismutase activity using standard NBT method. The complex has SOD activity with the IC(50) value of 1.32 μM.     

Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding

The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.     

Synthesis and copper(II)-binding properties of the N-terminal peptide of human alpha-fetoprotein.

The N-terminal native sequence tripeptide of alpha-fetoprotein, L-threonyl-L-leucyl-L-histidine N-methylamide, was synthesized and its interaction with Cu(II) ions was investigated by potentiometric titration at 25 degrees C in 0.15 M-NaCl and by visible-absorption, e.p.r. and n.m.r. spectroscopy. Analyses of the results in the pH range 4-10 indicated the presence of multiple complex species in solution: MHL, MH-2L, MHL2, ML2 and MH-1L2, where M, H and L represent metal ion, proton and ligand anion respectively. Only the species MH-2L and MH-1L2 are present in significant amounts at physiological pH. The results of the visible-absorption spectroscopy are consistent with the findings of species distribution that MH-2L is the major complex species detected above physiological pH that has the spectral characteristics of lambda max. = 523 nm and epsilon max. = 98 M-1.cm-1. The nine superhyperfine lines in e.p.r. spectra of the major species MH-2L strongly support the co-ordination of four nitrogen atoms by Cu(II). Both 1H- and 13C-n.m.r. studies suggest that the species MH-2L is a square-planar complex. The results from the equilibrium-dialysis experiments showed that this peptide is able to compete with albumin for Cu(II) ions. At equimolar concentrations of albumin and the peptide, about 52% of the Cu(II) was bound to the peptide. The possibility that alpha-fetoprotein plays an important role as the Cu(II)-transport protein in fetal life is discussed.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

Cu(I) binding properties of a designed metalloprotein

The Cu(I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an α-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu(I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu(I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600 nm emission. It was further found that the exogenous reductant tris(2-carboxyethyl)phosphine (TCEP) can competitively inhibit the binding of Cu(I) to the low affinity site of the peptide, but does not interact with Cu(I) clusters.     

Facile synthesis of a dimeric dipyrrole-polyamide and synergetic DNA-cleaving activity of its Cu(II) complex

Inspired by the potent DNA-cleaving activity of the Cu(II) complex of monopyrrole-polyamide dimer 1 (i.e., 1@Cu(2+)), we designed a new dimeric dipyrrole-polyamide analog 2 with the aim to optimize the catalytic activities of the metal complexes of this type of polypyrrole-polyamides. Compound 2 was prepared in 50% yield from the reaction of 1-methyl-4-[(1-methyl-4-nitro-1H-pyrrole-2-carbonyl)-amino]-1H-pyrrole-2-carboxylic acid with 2,2'-(ethane-1,2-diylbis(oxy))diethanamine, and fully characterized on the basis of NMR ((1)H and (13)C), MS (ESI and HR) and IR. Spectrophotometric titration, ESI-MS and conductivity measurements indicated that compound 2 formed a 1:1 complex with Cu(2+) ion (i.e., 2@Cu(2+)). Agarose gel electrophoresis studies indicated that 2@Cu(2+) was capable of efficiently converting pBR322 DNA into open circular and linear forms under physiological conditions, most probably via an oxidative mechanism. Its overall catalytic activity was estimated to be at least 30-fold higher than that of 1@Cu(2+). The fact that the cleaving activities of these Cu(II) complexes parallel, exactly, their binding affinities, raises the possibility that the cleaving activities of polypyrrole-polyamide derivatives of the type can be regulated by the binding affinities.     

Copper(II) complexes of tridentate pyridylmethylethylenediamines: role of ligand steric hindrance on DNA binding and cleavage

Copper(II) complexes of three linear unsymmetrical tridentate ligands viz. N-methyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L1), N,N-dimethyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L2) and N,N-dimethyl-N'-((6-methyl)pyrid-2-ylmethyl)ethylenediamine (L3) have been isolated and characterized by elemental analysis, electronic absorption and EPR spectroscopy and cyclic and differential pulse voltammetry. Of these complexes [Cu(L2)Cl2] and [Cu(L3)Cl2] have been structurally characterized by X-ray crystallography. The [Cu(L2)Cl2] complex crystallizes in the monoclinic space group P2(1)/n with a=11.566(2) A, b=7.369(1) A, c=15.703(3) A, alpha=90 degrees , beta=109.68(8) degrees , gamma=90 degrees and Z=4 while [Cu(L3)Cl2] crystallizes in the triclinic space group P1 with a=9.191(2) A, b=12.359(3) A, c=14.880(3) A, alpha=79.61(13) degrees , beta=86.64(13) degrees , gamma=87.28(8) degrees and Z=2. The coordination geometries around copper (II) in these two complexes are best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). The distorted CuN3Cl basal plane in them is comprised of three nitrogen atoms of the meridionally coordinated ligand and a chloride ion and the axial position is occupied by the other chloride ion. The interaction of these complexes with Calf Thymus DNA (CT DNA) has been studied by using absorption, emission and circular dichroic spectral methods, thermal denaturation studies, viscometry and cyclic and differential pulse voltammetry. A strong blueshift in the ligand field band and a redshift in the ligand based bands of the copper(II) complexes on binding to DNA imply a covalent mode of DNA binding of the complexes, which involves coordination of most possibly guanine N7 nitrogen of DNA to form a CuN4 chromophore. This is supported by studying the interaction of the complexes with N-methylimidazole (N-meim), guanosine monophosphate (GMP), adenosine monophosphate (AMP) and cytidine (cytd) by ligand field and EPR spectral methods, which indicate the formation of a CuN4 chromophore only in the case of the more basic N-meim and GMP. The DNA melting curves obtained in the presence of copper(II) complexes reveal a monophasic and irreversible melting of the DNA strands and the high positive DeltaTm values (12-21 degrees C) also support the formation of strong Cu-N bonds by the complexes with DNA, leading to intra- and/or interstrand crosslinking of DNA. Competitive ethidium bromide (EthBr) binding studies show that the L2 and L3 complexes are less efficient than the L1 complex in quenching EthBr emission, which is consistent with their forming DNA crosslinking preventing the displacement of the DNA-bound EthBr. A very slight decrease in relative viscosity of DNA is observed on treating the L1 and L2 complexes with CT DNA; however, a relatively significant decrease is observed for the L3 complex suggesting that the length of the DNA fiber is shortened. DNA cleavage experiments show that all the complexes induce the cleavage of pBR322 plasmid DNA, the complex of L1 being more efficient than those of sterically hindered L2 and L3 ligands.     

Effect of a conjugated acridine moiety on the binding and reactivity of Cu(II)[9-acridinylmethyl-1,4,7-triazacyclononane] with DNA

The DNA binding orientation and dynamic behavior of Cu(II) complexes of 1,4,7-triazacyclononane ([9]aneN(3)), 1, and an acridine conjugate, 2, were investigated by DNA fiber EPR (EPR=electron paramagnetic resonance) spectroscopy. Crystal and molecular structure of 2 were determined by X-ray diffraction. It has been shown that 1 binds to DNA in two different modes at room temperature; one species is rapidly rotating and the other is immobilized randomly on the DNA. The introduction of acridine to [9]aneN(3) fixed the [Cu([9]aneN(3))](2+) moiety of 2 in two different environments on the DNA: the g(mid R:mid R:) axis of one species (g( parallel)=2.26) is aligned perpendicularly to the DNA fiber axis whereas that of the other (g( parallel)=2.24) aligns<90 degrees with the DNA fiber axis. The different DNA binding structures of 1 and 2 are reflected also in their different efficiencies of DNA cleavage; 2 was found to be more effective both in oxidative and hydrolytic cleavage reactions.     

Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded alpha-helical coiled-coil toward a prototype for a metalloenzyme.

We previously reported the IZ-3adH peptide, which formed a triple-stranded coiled-coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ-3aH, having a new metal binding specificity. The IZ-3aH peptide was found to bind Cu(II) and Zn(II) and form a triple-stranded coiled-coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K(d) were 9 microm for Zn(II) and 10 microm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H(2)O.     

Specific cleavages of DNA by ascorbate in the presence of copper ion or copper chelates

The DNA-cleavage specificity of ascorbate in the presence of copper ion is analyzed with end-labeled pBR322 DNA fragments. The nonenzymatic reaction of Cu(II)/ascorbate and DNA shows certain degrees of cleavage preference toward purine-containing short segments in the labeled DNA under mild conditions (at 0 degrees C and 10 min). The segments of pyrimidine clusters are least susceptible to cleavage. The DNA scission cannot be detected in the absence of metal ions, and is greatly diminished in the presence of EDTA and metal-chelating peptide. It is more specific than the nuclease-like scission activity induced by cuprous-phenanthroline complex. This scission activity in relation to the antiviral and antitumor activities of vitamin C reported in the literature deserves a crucial consideration.     

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II).Gly-Gly-His-derived metallopeptides

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II).Gly-Gly-His-derived metallopeptides containing D- vs. L-amino acid substitutions in the first peptide position. This examination included studies of Cu(II).D-Arg-Gly-His and Cu(II).D-Lys-Gly-His for comparison to metallopeptides containing L-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II).D/L-Pro-Gly-His and Cu(II).D/L-Pro-Lys-His. Results indicated that L-Arg/Lys to D-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA, whereas the replacement of L-Pro by D-Pro in Cu(II).L-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness observed between the D- vs. L-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the epsilon-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II).Xaa-Xaa'-His metallopeptides with DNA are also discussed.     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

Isolation and two-dimensional 1H-NMR of peptide [1-24] of dog serum albumin and studies of its complexation with copper and nickel by NMR and CD spectroscopy

The NH2-terminal peptide fragment [1-24] of dog serum albumin was obtained by controlled peptic digestion of the protein. The peptide was purified to homogeneity by gel filtration and ion-exchange chromatography. The NMR assignments of the protons of the individual amino acid residues were made by using two-dimensional correlation matrix, spin-decoupling experiments and analysis of the titration curves. The polypeptide itself has a random-coil conformation. There is a conformational change as a function of pH, but it does not arise from any direct involvement of the amino acid side chains. Complexation of the peptide fragment with Ni(II) and Cu(II) has been investigated by NMR and CD. The Ni(II) complex is in slow exchange with the free ligand on the NMR time scale. The complexation involves the alpha-NH2, three deprotonated amide nitrogens of Ala-2, Tyr-3 and Lys-4 residues. The phenolate oxygen of Tyr-3 is not involved in the metal binding; however, an interaction between the aromatic ring and the metal ion is likely. The CD results of Cu(II)-binding to this peptide suggest that the complexation takes place from the terminal NH2 and step by step to three deprotonated amide nitrogens. There is no major conformational change of the peptide fragment upon complexation.